Collagen type ii oriented three-dimensional extracellular matrix and methods of designing and using same

Abstract

The application discloses a type II collagen-oriented three-dimensional extracellular matrix and a design method and application thereof. The application utilizes molecular structure analysis and molecular dynamics simulation, and is accurate in designing chiral materials aiming at the spatial structure of type II collagen, so that the material surface is highly spatially complementary to the active site of the protein, and the conformation regulation and specific recognition functions thereof are verified by calculation and experiment. The preparation process of the application can systematically regulate the micro-nano structure, chiral parameters and surface activity of the material, and realizes efficient unification of the physicochemical properties and biological functions of the material. The three-dimensional extracellular matrix of the application can guide the selective adsorption of type II collagen on the material surface, exposure of the ideal conformation and activation of the functional domain, realize spatial fine locking of the protein molecules, improve the activation efficiency of the signal path, and has wide application prospects in the field of tissue regeneration and repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and in particular relates to a type II collagen-guided three-dimensional extracellular matrix, its design method, and its application. Background Technology

[0002] Cellular recognition and response to the external environment play a crucial role in tissue regeneration and repair. The extracellular matrix (ECM), as a scaffold for cells, not only provides mechanical support but also finely regulates cell proliferation, differentiation, and migration through interactions with receptors such as cell surface adhesion proteins and integrins. Numerous studies have shown that these molecular recognition processes exhibit high stereoselectivity, and the spatial structural complementarity between adhesion proteins and receptors directly affects signal transduction efficiency and downstream biological responses. However, current designs of tissue-engineered ECM materials largely focus on increasing the adsorption of adhesion proteins on the material surface or introducing specific functional groups to enhance cell adhesion, which has the following limitations: (1) Lack of fine-grained control over protein spatial conformation: Existing material design focuses on the quantity of protein adsorption, neglecting the high spatial resolution of geometric complementarity control between proteins and materials, and cannot effectively reproduce the precision of natural spatial complementarity between receptors and ligands. Taking RGD-modified scaffolds as an example, although active groups are introduced, the spatial exposure angle of RGD and its matching with the receptor are not ideal, resulting in limited receptor activation efficiency.

[0003] (2) Poor spatial selectivity: Most matrix materials attract proteins only through chemical adsorption or electrostatic interaction, and cannot selectively induce stereoisomeric rearrangements that are beneficial to cell activation, which can easily lead to protein denaturation or loss of function. Materials such as molecular imprinting have problems such as complex preparation and difficulty in ensuring biocompatibility, and their effect on regulating higher-order protein structures is limited.

[0004] (3) Weak dynamic regulation capability: Existing technologies are mostly static adsorption or simple biomimetic grafting, which are difficult to dynamically regulate molecular events of protein adsorption, desorption and conformation, and lack active regulation means for adsorption process and spatial matching.

[0005] (4) Limited improvement in signal activation efficiency: Existing technologies can usually only improve the total amount of protein adsorbed, and have limited improvement on the activation rate of downstream signals.

[0006] Therefore, there is an urgent need to develop three-dimensional extracellular matrix materials that combine high spatial resolution structural regulation and excellent biocompatibility to achieve efficient tissue regeneration or repair. Summary of the Invention

[0007] This invention utilizes suitable chiral amino acids and fatty acids for synergistic self-assembly, thereby achieving the large-scale preparation of three-dimensional matrix materials with well-defined chiral mirror structures. The preparation process of this invention can systematically control the micro / nano structure, chiral parameters, and surface activity of the materials, achieving a highly efficient unification of the materials' physicochemical properties and biological functions. Specifically, this invention includes the following:

[0008] In a first aspect, the present invention provides a method for designing a type II collagen-guided three-dimensional extracellular matrix, comprising the following steps: (1) Construct a three-dimensional structural model of type II collagen for interface analysis, and establish a chiral surface unit library based on the geometric features of the receptor binding pocket of type II collagen. The chiral surface unit library contains amino acid units and fatty acid units. (2) Connect the three-dimensional structural model with the chiral surface primitive library, calculate the shape complementarity and interface energy, retain the top-ranked chiral surface primitive combinations based on the calculation results, construct a machine learning model, input elements into the machine learning model and output a comprehensive score, retain the chiral surface primitive combinations with the highest comprehensive scores among the top-ranked chiral surface primitive combinations, the elements include geometric features, physicochemical features and dynamic priors; (3) Perform all-atom molecular dynamics simulation on the chiral surface unit combinations with the highest comprehensive scores to determine the chiral surface unit combinations that meet the evaluation criteria, and use the chiral surface unit combinations that meet the evaluation criteria to perform self-assembly to obtain the type II collagen-guided three-dimensional extracellular matrix.

[0009] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix of the present invention, in step (1), the triple helix structure of type II collagen is targeted, high-resolution crystal structure is obtained from protein database, or prediction software is used for constrained prediction to generate ≥20 candidate conformations, and the prediction confidence level pLDDT≥80 and side chain exposure are used as screening criteria. After standardization, a three-dimensional structural model of type II collagen for interface analysis is obtained.

[0010] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix of the present invention, in step (1), the amino acid includes L- / D-amino acid enantiomers, enantiomeric glycine or amino acid derivatives thereof, and the fatty acid includes fatty acids containing 12 to 24 carbon atoms in their carbon chain or fatty acid derivatives thereof.

[0011] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix according to the present invention, in step (1), the geometric features of the receptor binding pocket of type II collagen include chiral curvature κ, pitch p, radius r and triple helix surface exposure.

[0012] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix of the present invention, in step (2), the top-ranked chiral surface motif combinations are retained as screening criteria with shape complementarity ≥0.60 and interface energy in the top 20%.

[0013] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix of the present invention, the geometric features include κ, p, r and triple helix surface exposure, the physicochemical features include hydrophobic / charged plaque area and hydrogen bond donor-acceptor density, and the kinetic priors include interfacial contact probability in short-range molecular dynamics.

[0014] In some embodiments, according to the design method of type II collagen-guided three-dimensional extracellular matrix of the present invention, in step (3), the evaluation index includes: adsorption free energy E ads Interface stability, conformational advantages, and preparation feasibility.

[0015] In a second aspect, the present invention provides a type II collagen-guided three-dimensional extracellular matrix, which is obtained by the design method described in the first aspect of the present invention.

[0016] In some embodiments, the type II collagen-guided three-dimensional extracellular matrix according to the present invention comprises a chiral surface unit assembly for preparing it, wherein D-glutamic acid and stearic acid are used.

[0017] A third aspect of the present invention provides a composition for tissue regeneration and repair comprising the three-dimensional extracellular matrix and type II collagen described in the second aspect of the present invention.

[0018] A fourth aspect of the invention provides the use of the three-dimensional extracellular matrix according to the second aspect of the invention or the composition according to the third aspect in the preparation of tissue regeneration and repair materials.

[0019] This invention achieves, for the first time, a high degree of spatial complementarity between three-dimensional materials and target proteins, enabling selective and targeted conformational regulation of proteins at the molecular level (such as significantly enhancing the β-sheet content in secondary structures). This invention efficiently reproduces mirror-helical structures through self-assembly, possessing advantages such as standardized material preparation processes, large-scale reproducibility, and excellent physicochemical properties. It achieves precise prediction of protein molecular dynamics and utilizes molecular dynamics and artificial intelligence screening, representing a fundamental breakthrough.

[0020] Furthermore, this invention enhances the bioactivity of tissue engineering scaffolds, applicable not only to osteochondrium but also to various extracellular matrix-related fields and the spatial regulation of various adhesion proteins. It provides a novel theoretical and methodological foundation for the development of tissue regeneration materials, fills a gap in the field of high-resolution structural regulation materials, improves the efficiency of tissue defect repair and the quality of functional regeneration, and supports the industrialization of regenerative medicine and high-end biomedical materials. Attached Figure Description

[0021] Figure 1 The scanning electron microscope results and appearance of the D-ECM are shown.

[0022] Figure 2 The Fourier transform infrared (FTIR) and X-ray diffraction (XRD) results are shown. A is the FTIR result, showing the formation of typical amide I and II absorption peaks in the D-ECM. B is the XRD result, showing that the D-ECM is a highly ordered mirror helix.

[0023] Figure 3 The results of CCK-8 assay show that D-ECM maintained a survival rate of over 90% for human chondrocyte precursor cells within 72 hours.

[0024] Figure 4 The results show the real-time monitoring of the adsorption kinetics of type II collagen (Col2a1) on D-ECM and PEG surfaces using a quartz crystal microbalance (QCM).

[0025] Figure 5 The content of Col2a1 secondary structures in D-ECM and PEG scaffolds is shown.

[0026] Figure 6 The exposure angles of the Col2a1 functional domains in D-ECM and PEG scaffolds are shown.

[0027] Figure 7 The distances between Col2a1 functional groups in D-ECM and PEG scaffolds are shown.

[0028] Figure 8 The activation levels of Itgα10 by D-ECM and PEG scaffolds are shown.

[0029] Figure 9 Arthroscopic observations of articular cartilage defect repair using D-ECM and control materials are shown.

[0030] Figure 10 The deposition and arrangement of type I and type II collagen fibers after articular cartilage defect repair using D-ECM and control materials are shown.

[0031] Figure 11The ICRS scores of D-ECM and control materials in articular cartilage defect repair are shown. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Design Methodology This invention utilizes molecular structure analysis and molecular dynamics simulation to precisely design chiral materials targeting the spatial structure of type II collagen, achieving a high degree of spatial complementarity between the material surface and the protein's active sites. Its conformational regulation and specific recognition functions are verified through both calculation and experimentation.

[0036] One aspect of the present invention provides a method for designing a type II collagen-guided three-dimensional extracellular matrix, comprising the following steps: (1) Construct a three-dimensional structural model of type II collagen for interface analysis, and establish a chiral surface unit library based on the geometric features of the receptor binding pocket of type II collagen. The chiral surface unit library contains amino acid units and fatty acid units. (2) Connect the three-dimensional structural model with the chiral surface primitive library, calculate the shape complementarity and interface energy, retain the top-ranked chiral surface primitive combinations based on the calculation results, construct a machine learning model, input elements into the machine learning model and output a comprehensive score, retain the chiral surface primitive combinations with the highest comprehensive scores among the top-ranked chiral surface primitive combinations, the elements include geometric features, physicochemical features and dynamic priors; (3) Perform all-atom molecular dynamics simulation on the chiral surface unit combinations with the highest comprehensive scores to determine the chiral surface unit combinations that meet the evaluation criteria, and use the chiral surface unit combinations that meet the evaluation criteria to perform self-assembly to obtain the type II collagen-guided three-dimensional extracellular matrix.

[0037] In this invention, "Type II collagen-guided three-dimensional extracellular matrix" refers to the ability of the three-dimensional extracellular matrix to guide or regulate the selective adsorption, ideal conformation exposure, and functional domain activation of Type II collagen on the material surface.

[0038] In a preferred embodiment, the design method of the present invention further includes the steps of characterizing the structure of the three-dimensional extracellular matrix and detecting biocompatibility. In this invention, the characterization is not particularly limited and can be performed using known methods and equipment, such as, but not limited to: observing the micro / nano helical structure of the material surface by SEM; analyzing hydrogen bonds, hydrophobic interactions, and secondary structure characteristic peaks by FTIR; and verifying ordered stacking and mirror correlation by XRD. Known methods and equipment can be used to detect biocompatibility, such as, but not limited to: detecting the viability of human chondrocytes or mesenchymal stem cells by CCK-8 assay, with a survival rate of ≥90%, demonstrating that the material possesses good biocompatibility.

[0039] In a preferred embodiment, the design method of the present invention further includes verifying the function of the three-dimensional extracellular matrix, wherein the verification is not particularly limited and can be performed using known methods and equipment, such as, but not limited to: using QCM to detect the adsorption rate and saturation curve of Col2a1 in real time; combining FTIR and molecular dynamics simulations to reveal the triple helix retention rate and secondary structure changes (e.g., but not limited to, changes in β-sheet content) of Col2a1 after adsorption on the material surface; determining the spatial angle and spacing between key functional domains of Col2a1 and the main chain by FRET to confirm its favorable exposure on the material surface; and using ELISA to analyze the activation level of Itgα10 by the three-dimensional extracellular matrix.

[0040] In step (1) of the present invention, the specific steps for constructing the three-dimensional structure model of type II collagen for interface analysis are not particularly limited. It can be obtained from a known protein database or screened by prediction software. For example, with the triple helix structure of type II collagen as the target, a high-resolution crystal structure can be obtained from a protein database, or a prediction software can be used to perform constrained prediction to generate ≥20 candidate conformations. The prediction confidence level pLDDT≥80 and the side chain exposure are used as screening criteria. After standardization, the three-dimensional structure model of type II collagen for interface analysis is obtained.

[0041] In step (1) of the present invention, the amino acid includes L- / D-amino acid enantiomers, enantiomerless glycine or amino acid derivatives thereof, and the fatty acid includes fatty acids containing 12 to 24 carbon atoms in their carbon chain or fatty acid derivatives thereof.

[0042] In this invention, the L-configured amino acids include: L-alanine (L-Ala), L-valine (L-Val), L-leucine (L-Leu), L-isoleucine (L-Ile), L-proline (L-Pro), L-methionine (L-Met), L-tryptophan (L-Trp), L-phenylalanine (L-Phe), L-serine (L-Ser), L-threonine (L-Thr), L-cysteine ​​(L-Cys); L-tyrosine (L-Tyr), L-asparagine (L-Asn), L-glutamine (L-Gln), L-lysine (L-Lys), L-arginine (L-Arg), L-histidine (L-His), L-aspartic acid (L-Asp), and L-glutamic acid (L-Glu).

[0043] In this invention, the D-configured amino acids include: D-alanine (D-Ala), D-valine (D-Val), D-leucine (D-Leu), D-isoleucine (D-Ile), D-proline (D-Pro), D-methionine (D-Met), D-tryptophan (D-Trp), D-phenylalanine (D-Phe), D-serine (D-Ser), D-threonine (D-Thr), D-cysteine ​​(D-Cys); D-tyrosine (D-Tyr), D-asparagine (D-Asn), D-glutamine (D-Gln), D-lysine (D-Lys), D-arginine (D-Arg), D-histidine (D-His), D-aspartic acid (D-Asp), and D-glutamic acid (D-Glu).

[0044] In this invention, the fatty acids containing 12 to 24 carbon atoms in the carbon chain include, but are not limited to, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoic acid, cis-9-tetradecenoic acid, cis-9-hexadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, cis-11-eicosenoic acid, cis-13-docosaenoic acid, cis-15-docosaenoic acid, cis,cis-9,12-octadecadienoic acid, cis-6,9,12-octadectrienoic acid, all-cis-5,8,11,14-eicosatetraenoic acid, all-cis-9,12,15-octadectrienoic acid, all-cis-5,8,11,14,17-eicosapridenic acid, and all-cis-4,7,10,13,16,19-docosahexaenoic acid.

[0045] In this invention, amino acid derivatives and fatty acid derivatives are also included within the scope of protection. The term "amino acid derivative" as used herein refers to compounds obtained by chemically modifying one or more functional groups in the parent molecule of an amino acid. Chemical modification includes, but is not limited to, modification of the amino group, modification of the carboxyl group, modification of the side chain functional groups, and modification of the main chain. Preferably, the modified amino acid derivative does not substantially alter the regulatory performance of the ECM on type II collagen or has improved regulatory performance or tissue regeneration and repair performance. Similarly, the term "fatty acid derivative" as used herein refers to compounds obtained by chemically modifying a straight-chain or branched, saturated or unsaturated fatty acid with 12 to 24 carbon atoms as the parent molecule. This chemical modification includes, but is not limited to, modification of the carboxyl group, modification of the carbon chain, modification of unsaturation, introduction of functional groups, and alteration of chain length. Preferably, the modified fatty acid derivative does not substantially alter the regulatory performance of the ECM on type II collagen or has improved regulatory performance or tissue regeneration and repair performance.

[0046] In step (1) of this invention, the geometric features of the receptor-binding pocket of type II collagen include chiral curvature κ, pitch p, radius r, and triple helix surface exposure. Preferably, the chiral curvature κ ranges from 0.01 to 0.50 nm. -1 The pitch p ranges from 10 to 200 nm, and the radius r ranges from 2 to 50 nm.

[0047] In step (2) of this invention, the top-ranked chiral surface unit combinations are retained based on the criteria of shape complementarity ≥ 0.60 and interfacial energy ranking in the top 20%. The geometric features include κ, p, r, and triple helix surface exposure, the physicochemical features include hydrophobic / charged patch area and hydrogen bond donor-acceptor density, and the kinetic priors include the interfacial contact probability in short-range molecular dynamics.

[0048] In step (3) of this invention, the evaluation index includes: adsorption free energy E ads The interface stability, conformational advantages, and preparation feasibility are all considered. Preferably, the adsorption free energy E... ads ≤-25 kcal•mol -1 The interface stability includes a Cα RMSF ≤ 1.5 Å for type II collagen interfaces and an interface contact duration ≥ 60%. The conformational advantages include a triple helix integrity retention rate of ≥ 90% for type II collagen and a relative increase in the exposure of key binding domains of ≥ 15%. The preparation feasibility includes the corresponding κ, p, and r falling within a controllable preparation window.

[0049] In this invention, chiral amino acids and long-chain fatty acids serve as key building blocks, achieving a three-dimensional chiral geometrically complementary structure through synergistic self-assembly. Reducing any component (e.g., using only D-glutamic acid or only octadecanestearic acid) significantly decreases the material's self-assembly capability, reduces the probability of spontaneously forming ordered mirror structures, and results in a lack of a high spatial resolution protein recognition surface, hindering effective spatial matching of target proteins. Experiments show that using only a single component often leads to non-specific aggregation or low-order structures, significantly reducing adsorption capacity and conformational regulation effects, with integrin signal activation efficiency less than 50% of that of the intact system.

[0050] In a preferred embodiment, the self-assembly of the present invention includes adding a crosslinking agent (e.g., but not limited to, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, etc.) to a neutral buffer system (e.g., but not limited to PBS), allowing the screened chiral units to undergo a coupling reaction for 5-20 h (preferably 6-18 h, more preferably 7-16 h, further preferably 8-14 h, more preferably 8-12 h, e.g., 8, 9, 10, 11, 12 h), removing small molecule impurities, and then freeze-drying to obtain a precursor powder. The precursor, after redispersing in the buffer system, can be controllably self-assembled into a D-ECM, forming a three-dimensional porous network structure with regular helical grooves. The method for removing small molecule impurities is not particularly limited and can be carried out by known methods, such as, but not limited to, dialysis (MWCO 1-5 kDa, 20-60 h).

[0051] In another preferred embodiment, the self-assembly of the present invention includes dissolving stearic acid in a solvent (e.g., but not limited to an aqueous ethanol solution with a volume ratio of 1:1), adding a pH adjuster (e.g., but not limited to MES) to adjust the pH to 5.5-6.5 (preferably 5.6-6.4, more preferably 5.7-6.3, even more preferably 5.8-6.2, e.g., 5.8, 5.9, 6, 6.1, 6.2), sequentially adding a crosslinking agent (e.g., but not limited to EDC, NHS, where the molar ratio of stearic acid:EDC:NHS can be 1:(1-2):(1-2)) under ice bath conditions, and magnetically stirring for 10-30 min (preferably 12-28 min, even more preferably 12-26 min, even more preferably 12-24 min, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) (min) Activate the carboxyl group; pre-dissolve D-glutamic acid in a small amount of aqueous solvent (e.g., deionized water, distilled water, ultrapure water, reverse osmosis water, etc.) with pH 7-8, slowly add it dropwise to the reaction solution, adjust the pH to 6.6-7.0, and react at 30-40℃ (preferably 31-39℃, even more preferably 32-38℃, such as 32, 33, 34, 35, 36, 37, 38℃) for 5-20 h (preferably 5-19 h, even more preferably 5-18 h, more preferably 5-17 h, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 h); dialyze the reaction solution with MWCO 1-5 kDa for 20-60 h (every 6-8 kDa). (h water change), freeze-dry to obtain a white porous precursor; disperse the precursor in a buffer system with pH 7-8 (e.g., but not limited to PBS), and self-assemble at 25-37℃ (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37℃) for 2-24 h (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 h) to obtain a three-dimensional D-ECM hydrogel.

[0052] Type II collagen-guided three-dimensional extracellular matrix In one aspect, the present invention provides a type II collagen-guided three-dimensional extracellular matrix obtained by the design method described in the present invention.

[0053] In a preferred embodiment, the chiral surface unit assembly used to prepare the three-dimensional extracellular matrix comprises or is composed of D-glutamic acid and stearic acid, preferably comprising or is composed of D-glutamic acid and octadecanestearic acid.

[0054] The three-dimensional extracellular matrix of this invention is highly spatially complementary to the target protein, enabling selective and targeted conformational regulation of the protein at the molecular level (such as significantly enhancing the β-sheet or triple helix content in the secondary structure). It has high signal activation efficiency and can efficiently activate the integrin signaling pathway (e.g., but not limited to Itgα10) (with an improvement of up to 30%), which is significantly superior to traditional adsorption or surface chemical modification materials.

[0055] Composition In one aspect, the present invention provides a composition for tissue regeneration and repair, comprising the three-dimensional extracellular matrix and type II collagen described herein. The composition of the present invention can guide the selective adsorption, ideal conformational exposure, and functional domain activation of adhesion proteins on material surfaces, achieving precise spatial locking of protein molecules based on the principle of chiral geometric complementarity, thereby improving the activation efficiency of signaling pathways.

[0056] use One aspect of the present invention provides the use of the type II collagen-guided three-dimensional extracellular matrix or composition described herein in the preparation of tissue regeneration and repair materials.

[0057] In this invention, tissue regeneration and repair materials include, but are not limited to, functionalized hydrogels, nanofiber scaffolds, and 3D-printed scaffolds. These materials may also contain proteins (or growth factors) and / or cells (such as stem cells, fibroblasts, chondrocytes, etc.).

[0058] In this invention, tissue regeneration and repair materials can be used for defects or damage to bone tissue or skin tissue, including but not limited to.

[0059] The present invention further provides a method for regulating cell proliferation, differentiation and migration in vitro, comprising the step of contacting and culturing cells with the aforementioned three-dimensional extracellular matrix or composition, wherein the cell surface contains integrins.

[0060] This invention also provides a method for in vitro regulation of cellular integrin signal activation levels, comprising the steps of contacting and culturing cells with the aforementioned three-dimensional extracellular matrix or composition, wherein the regulation specifically refers to upregulating cellular integrin signal activation levels. The upregulation of integrin signal activation levels can be determined using methods known in the art, including but not limited to flow cytometry using conformation-specific antibodies such as PAC-1 antibody and 12G10 antibody; it can also be determined by detecting the activation status of downstream signaling proteins of integrins (e.g., focal adhesion kinases, Src family kinases, Paxillin, etc.); or, the upregulation of integrin signal activation levels can be determined by detecting the cellular functional phenotype regulated by integrin signals, including but not limited to cell spreading assays, cell migration / invasion assays, scratch healing assays, and Transwell migration / invasion assays, to determine the cellular behavior regulated by activated integrin signals.

[0061] The cell types involved in this invention are not particularly limited, and examples include, but are not limited to, stem cells (dental pulp stem cells, human umbilical cord stem cells, bone marrow stem cells or embryonic stem cells, etc.), epithelial cells, osteocytes, etc.

[0062] Example 1 This embodiment illustrates a method for designing a type II collagen-guided three-dimensional extracellular matrix.

[0063] 1. Spatial complementary chiral structure analysis of targeting adhesion proteins Protein structure acquisition and prediction: The triple helix structure of human type II collagen Col2a1 was targeted, and its high-resolution crystal structure was obtained from the PDB database. If the structure was missing or the resolution was insufficient, AlphaFold2 was used for constrained prediction, generating ≥20 candidate conformations. Prediction confidence (pLDDT ≥ 80) and side chain exposure were used as screening criteria. After standardization, a three-dimensional structural model of type II collagen suitable for interface analysis was obtained.

[0064] Based on the geometric characteristics of the receptor-binding pocket in type II collagen, a chiral motif library composed of enantiomers of natural amino acids and C12-C24 fatty acids was established. The amino acids were preferentially selected from those with exposed outer layers, and the fatty acids were preferentially selected from 10 representative fatty acids that modulate the hydrophobic chain length and curvature. The geometric characteristics of the receptor-binding pocket in type II collagen include chiral curvature κ, pitch p, radius r, and triple helix surface exposure. This motif library achieves tunable complementarity between the surface helical grooves and the type II collagen receptor pocket by adjusting the spatial conformation of the amino acids and the chain length / unsaturation of the fatty acids.

[0065] 2. Deep Learning Prediction and Coarse Screening Scoring The three-dimensional structural model of type II collagen was individually docked with a chiral primitive library, and the Rosetta platform was used for interface construction and energy optimization. Shape complementarity (Sc) and interfacial energy ΔE were calculated. Rosetta And with Sc≥0.60 and ΔE Rosetta The top 20% of combinations advance to the next round of selection. Simultaneously, a composite machine learning model is constructed, with input factors including: geometric features (κ, p, r, triple helix surface exposure); physicochemical features (hydrophobic / charged plaque area, hydrogen bond donor-acceptor density); and kinetic priors (interfacial contact probability in short-range molecular dynamics). The output is a comprehensive score S. total The top 5-10% of combinations will be retained for molecular dynamics validation.

[0066] 3. Molecular dynamics simulations and elementary component screening Molecular dynamics conditions: Simulations were conducted on the GROMACS2020.2 platform using the CHARMM36 / OPLS-AA force field, TIP3P water model, and a 0.15 M NaCl environment. After energy minimization, NVT (300 K, 1 ns), and NPT (1 bar, 10 ns) equilibration, production phase simulations of ≥100 ns were performed, with each combination repeated three times.

[0067] Evaluation criteria: (1) Adsorption free energy E ads ≤ 25 kcal·mol -1 (2) Interface stability: Cα RMSF≤1.5Å, interface contact duration≥60%; (3) Conformation optimization: Col2a1 triple helix integrity retention≥90%, key binding domain exposure relatively increased≥15%; (4) Preparation feasibility: chiral parameters κ, p, r fall within the controllable synthesis window. Screening results: The optimal chiral amino acid-fatty acid combination was finally determined to be D-glutamic acid and octadecanestearic acid, which served as the core unit for material design.

[0068] 4. Self-assembly and preparation of matrix materials In a PBS buffer system (pH=7.4), an EDC / NHS-mediated amidation coupling reaction was used to couple selected amino acids to fatty acids. After 8–12 h of reaction, small molecule impurities were removed by dialysis (MWCO 3.5 kDa, 48 h), followed by freeze-drying to obtain the precursor powder. The precursor, after redispersion in PBS, underwent controlled self-assembly into a D-ECM, forming a three-dimensional porous network structure with regular helical grooves.

[0069] 5. Physicochemical and biological characterization Structural characterization: The micro / nano helical structure on the material surface was observed by SEM; hydrogen bonding, hydrophobic interactions, and secondary structure characteristic peaks were analyzed by FTIR; and ordered stacking and mirror correlation were verified by XRD. Biocompatibility: The viability of human chondrocytes or mesenchymal stem cells was detected by CCK-8 assay, and the survival rate remained ≥90%, demonstrating that the material has good biocompatibility.

[0070] 6. Protein Adsorption and Conformation Regulation Experiments Kinetic monitoring: The adsorption rate and saturation curve of Col2a1 were monitored in real time using QCM. Structural rearrangement analysis: Combining FTIR and molecular dynamics simulations, the triple helix retention rate and secondary structure changes (such as changes in β-sheet content) of Col2a1 after adsorption on the material surface were revealed. Functional domain exposure detection: The spatial angle and spacing between key functional domains of Col2a1 and the main chain were determined by FRET to confirm its favorable exposure on the material surface. Signaling pathway validation: The activation level of integrin Itgα10 was analyzed using ELISA.

[0071] 7. Data Statistics and Comparative Experiments The prepared chiral three-dimensional ECM was compared with the traditional control material (polyethylene glycol PEG scaffold) in parallel experiments. The comparison indicators included: total Col2a1 adsorption and kinetic parameters; triple helix retention rate and conformation optimization degree; functional domain exposure angle and spacing; Itgα10 signal activation efficiency; and cartilage regeneration efficiency.

[0072] Example 2 This embodiment illustrates the process of preparing D-ECM using D-glutamic acid and octadecanestearic acid obtained from Example 1, as well as the characterization results of D-ECM.

[0073] 1. Reaction materials D-Glutamic acid: 0.45 g (3.1 mmol, purity ≥99%); Stearic acid (C18:0): 1.20 g (4.2 mmol, purity ≥98%); EDC·HCl: 1.00 g (5.2 mmol); NHS: 0.62 g (5.4 mmol); Anhydrous ethanol / deionized water: 25 mL each (volume ratio 1:1); Buffer system: MES 0.1 M (pH=6.0, for carboxyl activation); PBS 0.1 M (pH=7.4, for self-assembly).

[0074] 2. Preparation method (1) Activation: Dissolve stearic acid in ethanol / water (1:1, v / v), add MES to adjust pH to 6.0; under ice bath conditions, add EDC and NHS sequentially (stearic acid: EDC: NHS≈1:1.2:1.3, molar ratio), and magnetically stir for 15 min to activate the carboxyl group.

[0075] (2) Coupling: D-glutamic acid was pre-dissolved in a small amount of deionized water (pH=7.5) and slowly added dropwise to the reaction solution. The pH was adjusted to 6.6-7.0 in real time, and the reaction was carried out at 37℃ for 8-12 h.

[0076] (3) Purification and drying: The reaction solution was dialyzed with MWCO 3.5 kDa for 48 h (water was changed every 6-8 h), and then freeze-dried to obtain a white porous precursor (overall yield 68-82%).

[0077] (4) Self-assembly and shaping: The precursor was dispersed in PBS (pH=7.4) and self-assembly was induced under controlled temperature (25-37℃, 2-24h) to obtain a three-dimensional D-ECM hydrogel. PEG was used as a control group.

[0078] 3. Physicochemical and biological characterization The D-ECM material was characterized, and the SEM results are as follows: Figure 1 As shown in Figure A, the surface of the D-ECM material exhibits a uniform helical structure with a fiber diameter of approximately 57.55 ± 9.33 nm; the FTIR results are as follows. Figure 2 As shown in Figure A, D-ECM exhibits typical amide I / II bands with hydrogen bond network enhancement; XRD results are as follows. Figure 2 As shown in B, the ordered stacking peaks of D-ECM exhibit a correlation with mirror images; biocompatibility is as follows: Figure 3 As shown, the survival rate of CCK-8 cells (24 / 72 h) was ≥90% when assessed using human chondrocyte precursor cells / mesenchymal stem cells (MSCs), and no acute cytotoxicity was observed.

[0079] Example 3 This embodiment demonstrates the Col2a1 conformational regulation and Itgα10 signal activation capability of the D-ECM prepared in Example 2.

[0080] 1. Experimental Materials Protein: Human Col2a1 (purity ≥95%), working concentration 50-200 μg·mL -1 ; Substrate: D-ECM, PEG; Buffer: PBS or HEPES (pH=7.4), ionic strength 0.15 M; Temperature: 25-37℃ (consistent with self-assembly conditions); Repetition: n≥3 for each group, unless otherwise stated.

[0081] 2. Experimental methods and results (1) Adsorption kinetics (QCM) Method: A precursor material was thinly deposited onto a QCM wafer (gold electrode). A Col2a1 solution was introduced into a flow cell, and the frequency shift (Δf) and impedance (ΔD) were recorded in real time. The half-peak time t1 / 2 and the plateau value R were obtained by fitting. max .

[0082] Key results: The D-ECM group reached a plateau within ≤7 min (t1 / 2 was significantly smaller than that of the control), R max The initial affinity was higher than that of the PEG group (p<0.01), indicating higher initial affinity and steady-state adsorption capacity. Figure 4 ).

[0083] (2) Triple helix retention and secondary structure (FTIR / CD) Methods: FTIR was measured under dry / wet conditions after adsorption; far-ultraviolet characteristic peaks were monitored by CD, and the triple helix indicator ratio was calculated.

[0084] Key results: On the D-ECM surface, the triple helix retention of Col2a1 was approximately 21.3% higher than that of the PEG group. Figure 5 ).

[0085] (3) Functional strip exposure and orientation (FRET / geometric measurement) Methods: Site markers were performed on the neighborhood of the Col2a1 functional stripe, FRET efficiency was measured, and the axial orientation angle θ of the stripe relative to the principal axis was geometrically inverted. helix Distance from key sites.

[0086] Key findings: The D-ECM group showed a significant increase in functional band exposure, θ helix Increase by 17-22° ( Figure 6 The donor-receptor distance is 1.1 ± 0.2 nm, which is within the favorable recognition range. Figure 7 ).

[0087] (4) Receptor activation (ELISA / Cellular) Methods: Activation efficiency was verified by ELISA using Itgα10 ligand binding as the readout.

[0088] Key results: D-ECM increased Itgα10 activation by approximately 32.8% compared to PEG (n≥3, p<0.01, two-tailed t-test). Figure 8 ).

[0089] Statistical notes: Data are expressed as mean ± SD; after normality testing, two-tailed t-tests or one-way ANOVA (Tukey post-hoc comparisons) were used; significance threshold p < 0.05.

[0090] Example 4 This example illustrates the activation of Itgα10 in the D-ECM prepared in Example 2 and the control material.

[0091] 1. Experimental Materials Control material: polyethylene glycol PEG scaffold; protein concentration, buffering and temperature conditions were the same as in Example 3.

[0092] 2. Experimental methods and results The experimental method is the same as in Example 3.

[0093] QCM results are as follows Figure 4 As shown: the PEG group reached the plateau time for a longer period, R max Lower; FTIR / CD results as follows Figure 5 As shown: triple helix retention improvement ≤7-9%, spectral peak changes are limited; FRET / geometric results are as follows. Figure 6 , 7 As shown: insufficient strip exposure, θ helix <55°, key site spacing deviates from the favorable recognition interval; Itgα10 activation level as... Figure 8 As shown: an increase of ≤10% indicates no significant or weakly significant difference.

[0094] The results show that traditional control materials mainly improve the adsorption of "quantity" but have difficulty in directionally stabilizing the triple helix and functional band orientation of Col2a1. The D-ECM of this invention achieves "adsorption-conformation-signal" coupling optimization through chiral geometric complementarity, thereby significantly enhancing the Itgα10 pathway and meeting the cartilage-directing requirements.

[0095] Example 5 This embodiment demonstrates the effects of D-ECM and control materials on promoting cartilage repair.

[0096] 1. Experimental Materials Materials: D-ECM hydrogel prepared in Example 2; control group: polyethylene glycol PEG scaffold; Experimental animals: SD rats, articular cartilage defect model (diameter 2 mm, depth 2 mm); Implantation method: Fill the defect area with an equal volume of D-ECM or control material; Observation period: 8 weeks post-surgery; Repetition: n≥6 animals per group.

[0097] 2. Experimental methods and results (1) Gross observation and imaging Methods: Postoperative fixed-point photography and arthroscopic observation were performed to assess the filling of the defect area.

[0098] Results: In the D-ECM group, the surface of the defect area was smooth and the color was consistent with the adjacent cartilage; the PEG residual defects were obvious and the filling was uneven. Figure 9 ).

[0099] (2) Histological staining (Sirius red) Methods: Joint samples were taken, decalcified, embedded, and sectioned. After Sirius Red staining, the deposition and arrangement of type I and type II collagen fibers were observed under a polarized light microscope.

[0100] Results: In the D-ECM group, type II collagen deposition was significant in newly formed tissue, with dense and uniformly distributed fibers; in the PEG group, type I collagen was predominant, with weaker type II collagen signals and loose, uneven fiber arrangement. Figure 10 ).

[0101] (3) ICRS score Methods: The International Cartilage Repair Society (ICRS) scoring system was used to comprehensively evaluate the repaired tissue based on dimensions such as surface smoothness, integration, and matrix type.

[0102] Results: At 8 weeks, the mean ICRS score in the D-ECM group was 9.2±1.1 points, significantly higher than that in the PEG group (6.3±0.9 points). Figure 11 ).

[0103] 3. Statistical Explanation All data are expressed as mean ± SD; after normality testing, one-way ANOVA and Tukey post-hoc test were performed; the significance threshold was set at p < 0.05.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for designing a type II collagen-guided three-dimensional extracellular matrix, characterized in that, Includes the following steps: (1) Construct a three-dimensional structural model of type II collagen for interface analysis, and establish a chiral surface unit library based on the geometric features of the receptor binding pocket of type II collagen. The chiral surface unit library contains amino acid units and fatty acid units. (2) Connect the three-dimensional structural model with the chiral surface primitive library, calculate the shape complementarity and interface energy, retain the top-ranked chiral surface primitive combinations based on the calculation results, construct a machine learning model, input elements into the machine learning model and output a comprehensive score, retain the chiral surface primitive combinations with the highest comprehensive scores from the top-ranked chiral surface primitive combinations, the elements include geometric features, physicochemical features and dynamic priors, and the top-ranked chiral surface primitive combinations are those with shape complementarity Sc ≥ 0.60 and interface energy ΔE Rosetta The top 20% of combinations, and the top 5 chiral surface primitive combinations in terms of overall score. 10% of the combinations; (3) Perform all-atom molecular dynamics simulation on the chiral surface unit combinations with the highest comprehensive scores to determine the chiral surface unit combinations that meet the evaluation criteria, and use the chiral surface unit combinations that meet the evaluation criteria to perform self-assembly to obtain the type II collagen-guided three-dimensional extracellular matrix.

2. The design method for type II collagen-guided three-dimensional extracellular matrix according to claim 1, characterized in that, In step (1), the triple helix structure of type II collagen is the target. High-resolution crystal structures are obtained from protein databases or prediction software is used for constrained prediction to generate ≥20 candidate conformations. The prediction confidence level pLDDT≥80 and side chain exposure are used as screening criteria. After standardization, a three-dimensional structural model of type II collagen for interface analysis is obtained.

3. The design method for type II collagen-guided three-dimensional extracellular matrix according to claim 1, characterized in that, In step (1), the amino acid includes L- / D-amino acid enantiomers, enantiomeric glycine or amino acid derivatives thereof, and the fatty acid includes fatty acids containing 12 to 24 carbon atoms in their carbon chain or fatty acid derivatives thereof.

4. The design method for type II collagen-guided three-dimensional extracellular matrix according to claim 1, characterized in that, In step (1), the geometric features of the receptor binding pocket include chiral curvature κ, pitch p, radius r, and triple helix surface exposure.

5. The design method for type II collagen-guided three-dimensional extracellular matrix according to claim 1, characterized in that, In step (2), the geometric features include chiral curvature κ, pitch p, radius r, and triple helix surface exposure; the physicochemical features include hydrophobic / charged patch area and hydrogen bond donor-acceptor density; and the kinetic priors include the interfacial contact probability in short-range molecular dynamics.

6. The design method for type II collagen-guided three-dimensional extracellular matrix according to claim 1, characterized in that, In step (3), the evaluation indicators include: adsorption free energy E ads Interface stability, conformational advantages, and preparation feasibility.

7. A type II collagen-guided three-dimensional extracellular matrix, characterized in that, Obtained by the design method described in any one of claims 1-6.

8. The type II collagen-guided three-dimensional extracellular matrix according to claim 7, characterized in that, The chiral surface building blocks used to prepare it include D-glutamic acid and stearic acid.

9. A composition for tissue regeneration and repair, characterized in that, It comprises the three-dimensional extracellular matrix and type II collagen as described in claim 7 or 8.

10. Use of the type II collagen-guided three-dimensional extracellular matrix according to claim 7 or 8, or the composition according to claim 9, in the preparation of tissue regeneration and repair materials.

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